The essential starting point for all our research is the continuing physicochemical characterization of a versatile class of NO-releasing prodrugs, the diazeniumdiolates. This fundamental chemical research program serves as a promising platform for designing improved biomedical research tools as well as potential clinical applications for them. As one example, current work is aimed at characterizing the mechanisms of NO versus HNO (nitroxyl, a newly identified bioeffector species) release in model diazeniumdiolates in collaboration with K. Miranda and D. Wink; a practical aim here is to design a "pure" HNO generator for therapeutic use. <BR>Glycosylated diazeniumdiolates have been shown to be reasonably stable at neutral or acidic pH but to undergo ready cleavage under catalysis by glycosidases; this finding has allowed us to design prodrugs useful for targeting NO to macrophages (collaboration with C. Bogdan). <BR>The PROLI/NO anion has shown particular promise for biomedical applications because of its favorable toxicological profile and the fact that its dissociation to NO is so rapid (half-life 2 seconds at pH 7.4 and 37<SUP>o</SUP>C) that the pharmacological effects can be effectively localized at the point of introduction into the body. But this sensitivity to decomposition has complicated various attempts to formulate it for biomedical use. We have been able during the past year to devise a successful general method for synthesizing O-protected derivatives of PROLI/NO for possible therapeutic use. In another specific application, we are exploring O-vinylated derivatives as non-toxic prodrugs for targeting NO to the liver and kidney (collaboration with J. Liu and M. Waalkes). <BR>Diazeniumdiolate anions of structure R<SUB>2</SUB>NN(O)=NO<SUP>-</SUP> have proven ideal for studying the reactivity of NO in aerobic aqueous media. Molecular NO can itself be used for this purpose, but the fact that it rapidly oxidizes to produce reactive nitrous acid (HONO) can greatly complicate interpretation of the results--were the reactions observed due to aerobic NO or to HONO? Conducting the study in well buffered media can of course be employed, but one must still be wary of transient local excesses of nitrite ion at low pH when the reactants are mixed. Diazeniumdiolate anions allow the researcher to circumvent this problem, because a basic amine moiety is generated at each and every site in the medium where an NO is released, ensuring that the pH in that microcosm cannot be lowered to activate the otherwise unreactive nitrite ion. This advantage of the simple R<SUB>2</SUB>NN(O)=NO<SUP>-</SUP> ions has been exploited to show that aerobic NO can induce nitrosative deamidation of asparagine and glutamine residues in peptides and proteins. Current work is aimed at determining the potential biological significance of this possible post-translational modification, whose mechanism differs in very fundamental ways from that of the established hydrolytic deamidation pathway. <BR>Work continues on other aspects of the chemistry and pharmacology of NO and the diazeniumdiolates, including those in which the -N(O)=NO<SUP>-</SUP> group is attached to polymers of interest in possible surgical and wound healing applications (collaboration with M. Kibbe, M. Meyerhoff, and several commercial organizations). <BR>Our major current focus is on O<SUP>2</SUP>-aryl diazeniumdiolates, agents designed to be activated for NO release by reaction with the abundant cellular nucleophile glutathione; with colleagues at several locations, we are vigorously pursuing efficacy and mechanisms studies of two lead compounds in this series, JS-K and PABA/NO (collaborations with P. Shami, A. Tari, T. Kiziltepe, K. Anderson, M. Malik, X. Ji, S. Yuspa, D. Townsend, and K. Tew), as promising anticancer agents. <BR>Considerable effort is also being devoted to synthesizing "NONO-NSAIDs", hybrid molecules that link aspirin or other non-steroidal antiinflammatory drugs to the diazeniumdiolate group for tests both as improved antiinflammatories and as agents for chemoprevention of cancer (collaboration with E. Knaus)